Neuronal CaMKK2 promotes immunosuppression and checkpoint blockade resistance in glioblastoma

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME) and is refractory to immune checkpoint blockade (ICB). Here, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a driver of ICB resistance. CaMKK2 is highly expressed in pro-tumor cells and is associated with worsened survival in patients with GBM. Host CaMKK2, specifically, reduces survival and promotes ICB resistance. Multimodal profiling of the TME reveals that CaMKK2 is associated with several ICB resistance-associated immune phenotypes. CaMKK2 promotes exhaustion in CD8+ T cells and reduces the expansion of effector CD4+ T cells, additionally limiting their tumor penetrance. CaMKK2 also maintains myeloid cells in a disease-associated microglia-like phenotype. Lastly, neuronal CaMKK2 is required for maintaining the ICB resistance-associated myeloid phenotype, is deleterious to survival, and promotes ICB resistance. Our findings reveal CaMKK2 as a contributor to ICB resistance and identify neurons as a driver of immunotherapeutic resistance in GBM.

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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
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MRI-based neuroimaging
Unprocessed scRNA-seq data has been uploaded to NCBI Gene Expression Omnibus ((https://www.ncbi.nlm.nih.gov/geo/) under data repository accession number https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE197879. The processed Seurat objects have also been made available through zenodo under record number https://zenodo.org/record/6654420. The data necessary to reproduce the graphs presented within this manuscript are provided in the Source Data file. The publicly available data used in this study were obtained from the GENT2 database http://gent2.appex.kr/gent2/, from the GBMseq portal http://gbmseq.org/, and from the Allen Brain Map https://portal.brain-map.org/atlases-and-data/rnaseq/human-m1-10x. The mm10 reference assembly is available through GenBank under accession code GCA_000001635.2 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/). The remaining data are available within the article, Supplementary Information or Source Data file.
Power analysis was not used to predetermine sample size. Sample sizes were instead determined based on historical sample sizes that were capable of detecting biologically significant differences for certain assays. If no historical data was available, pilot experiments were performed to determine the relative variability of the assay.
No data was excluded.
All experiments were successfully repeated at least twice.
Mice were randomly assigned to treatment groups within a given genotype. All experiments involving treatments were performed in-vivo and not in-vitro. For non-treatment, in-vivo or ex-vivo experiments, mice were randomly selected from age,sex, and genotype matched breeding cohorts.
Survival experiments were partially monitored by blinded veterinary staff. Veterinary staff were unable to monitor the experiments everyday so unblinded investigators assisted in survival monitoring.
Confocal microscopy images were acquired, and analyzed by a blinded co-author.
All other experiments were analyzed by unblinded investigators due to personnel shortages not allowing for availability of separate investigators for data acquisition and analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Antibodies used are noted in Supplemental Table 8.
Extensive validation was performed, and detailed in a separate manuscript (PMID: 34597618). Antibodies utilized in this manuscript were optimized by titrating to achieve a maximal staining index.
All cell lines were authenticated and tested by IDEXX Laboratories. Interspecies contamination was evaluated by species specific PCR and STR profiling.
All cell lines were confirmed to be mycoplasma negative by IDEXX Laboratories.
No commonly misidentified cell lines were used in this study.
Six-to eight-week-old C57BL/6J, LysMcre, Syn1cre, and CD45.1 mice were purchased from the Jackson Laboratory. CaMKK2-/-, Tg (Camkk2-EGFP)DF129Gsat reporter mice (CaMKK2-EGFP), and CaMKK2fl/fl mice were generously provided by Luigi Racioppi (Duke University). CaMKK2-/-, CaMKK2-EGFP and CaMKK2fl/fl mice have been previously validated. Animals were maintained under pathogen-free conditions at the Cancer Center Isolation Facility of Duke University Medical Center. Experiments were conducted on age and sex matched female mice between 8-12 weeks of age. Animals were maintained under pathogen-free conditions, in temperature and humidity controlled housing, with free access to food and water, under a 12-h light/dark cycle at the Cancer Center Isolation Facility of Duke University Medical Center.
This study did not include wild animals.
This study did not include specimens collected from the field.
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Duke University Medical Center.
Sample preparation is extensively detailed here (PMID: 34597618). IIn brief, tumor-bearing hemispheres were harvested on day 14 post tumor implantation. Tissue was transferred to a Dounce Tissue Homogenizer with 5 mLs of digestion cocktail containing 0.05 mg ml-1 LIberase DL (Roche), 0.05 mg ml-1 Liberase TL (Roche), 0.2 mg ml-1 DNase I (Roche) in HBSS with calcium and magnesium. A cell suspension was obtained after 5-10 strokes with the loose-fitting (A-size) pestle. The cell mixture was then incubated at 37°C for 15 min in a water bath to obtain a single-cell suspension. The single-cell suspension was then passed through a 70-µm filter. After centrifugation, the cells were resuspended in 1x RBC Lysis Buffer (Thermo Fisher Scientific) for 3 minutes. Myelin was removed from the sample with Percoll centrifugation. Samples were centrifuged and mixed with 30% Percoll (Sigma Aldrich) and centrifuged at 500g for 20 min at 18°C with no brake. The myelin layer and Percoll were then aspirated and the pellet was re-suspended in PBS before counting on an automated cell counter (Thermo Fisher Scientific).